Antimutagenic, antitumor and estrogen receptor binding activity of the rare plant Shortia galacifolia: An ethnobotanical and chemosystematic approach.

Objective
Shortia and other members of the Diapensiaceae family have ethnomedicinal history in both Eastern and Western hemispheres. Based on ethnopharmacological and chemosystematic evidence, pharmacological and toxicological bioassays were conducted on the rare plant Oconee Bell, Shortia galacifolia.


Materials and Methods
Extracts were examined in assays for antimutagenicity, antitumor and estrogen receptor (ER)-binding activity. Antitumor activity was assessed by the tumor induction assay (TiA), using Agrobacterium tumefaciens based on its ability to transform plant tissue. Antimutagenicity was examined using the Ames bacterial reverse mutation test. Recombinant human ERα and ERβ proteins were utilized to screen extracts for receptor selectivity.


Results
All concentrations of extracts inhibited A. tumefaciens-induced tumor formation on potato discs, with the mature rhizome extracts having the most marked inhibition. All three plant extracts significantly inhibited the formation of histidine-independent revertant colonies after exposure to the mutagen 2-aminoanthracene (2-AA) in the Ames Salmonella mutagenicity assay. In the ER binding assays, ERβ, but not ERα, displayed affinity for Shortia extracts.


Conclusion
Antitumor, ER binding and antimutagenic activities of S. galacifolia extracts were identified using rapid bench-top assays and warrant further investigations.


Introduction
What is the best approach to screening for phytomedicinals when ethnomedicinal knowledge is lacking? In such cases, an approach involving chemosystematics, also called pharmaphylogeny, may be useful (Cozzo, 2004;Pieroni and Vandebroek, 2007;Vick, 2011). Chemosystematics refers to a screening approach that focuses on specific chemicals, classes of chemicals or bioactivity within specific plant taxa (Atanasov et al., 2015;Pieroni and Vandebroek, 2007;Song et al., 2016).
The Diapensiaceae is one such family that may benefit from this approach and includes five genera: Shortia, Galax and Berneuxia, shown in Figure  1, Pyxidanthera and Diapensia (Ronblom and Anderberg, 2002). The family has a disjunct distribution, described in Figure 1, with members occurring in eastern North America and the Sino-Japanese Floristic Region (Ronblom and Anderberg, 2002). In the following paragraphs, we will present a chemosystematic rationale for examining Shortia galacifolia T. & G. for potential medicinal activity (see Table 1).
The plant that is the focus of this study, Shortia galacifolia, is indigenous to tiny isolated areas of the Southern Appalachian Mountains of North Carolina and the Jocassee Gorges of upstate South Carolina (Davies, 1955;Dunn and Jones, 1979). It is a rhizomatous, low-growing, evergreen plant with dark, glossy green leaves that turn reddish in the winter (Davies, 1955;Dunn and Jones, 1979). It blooms in early spring with a single bell-shaped white or pink flower on a slender peduncle 6-8 inches high as shown in Figure 1, giving rise to its common name, the Oconee Bell. This rare plant was collected for scientific identification in 1787 by André Michaux and later encountered in 1839 by Asa Gray in a Paris herbarium (Jenkins, 1946;Vivian, 1967).
A bit of ethnobotanical evidence was provided in Michaux's notes: that the Cherokee people inhabiting the Keowee area at the time Michaux collected his Shortia specimen were familiar with the plant and noted that it had a good taste and pleasing aroma (Jenkins, 1946;Vivian, 1967). Further ethnobotanical information on S. galacifolia has been lacking. Transmission of the ethnobotanical knowledge of this plant, like that of its related plant Galax, may have been negatively impacted by several factors including decades of warfare in the Keowee area before the development of the Cherokee writing system, the forced relocation of the Cherokee during the Indian Removal and the subsequent inundation of this historically important area in the creation of Lake Keowee (Cozzo, 2004;Pieroni and Vandebroek, 2007;Vick, 2011).
However, there is ethnomedicinal evidence for a different Shortia species from China, Shortia thibetica Decne. Franch is synonymous with Berneuxia thibetica Decne and Berneuxia yunnanensis H.L.Li, 岩筋菜, Yán Jīn Cài (shown in Figure 1; to avoid confusion we will use B. thibetica designation for the remainder of this article). B. thibetica is endemic to the southwest of China, including the Yunnan province, and is used in Traditional Chinese Medicine (TCM) as a cure for asthma, overstrain and cough (Wang et al., 1998). This work represents the first research, to our knowledge, of S. galacifolia in pharmacological and toxicological assays. Bioassays can provide a useful methodology for examination of plant extracts for antitumor, mutagenic and estrogen receptor (ER)-binding activity. The tumor induction assay (TiA) is based on the unique Agrobacterium tumefaciens characteristic of inserting a portion of its tumor-inducing (Ti) plasmid into wounded plants resulting in tumor-like growth, commonly known as Crown Gall disease (Galsky et al., 1981). Potential antitumor activity of plant extracts can be evaluated by measuring inhibition or stimulation of A. tumefaciens-induced tumor formation on potato discs treated with the plant extract (Galsky et al., 1981). Activity in the TiA has been correlated with antitumor activity in mammals (McLaughlin et al., 1998).
The Ames Salmonella mutagenic assay is a bacterial reverse mutation test which uses a histidine-dependent auxotrophic mutant of Salmonella typhimurium that lacks normal DNA repair mechanisms and cannot grow on histidine-free media (Mortelmans and Zeiger, 2000). Mutagenic changes are assessed by the number of colonies. Conversely, the degree to which a plant extract inhibits diagnostic mutagens can be used to evaluate antimutagenic and possibly anticarcinogenic potential (Friedman and Smith, 1984).
Chemosystematic analysis indicates that several members of Diapensiaceae display activities that may result from targeting the estrogen receptors (Garrett, 2003;Wang et al., 1998). ERα and ERβ can be utilized as a screening tool for extracts or individual chemicals with selective ER receptor modulatory (SERM) bioactivity without cell culture and eliminating any potential for crosstalk (Gray et al., 2004;Lackey et al., 2001). The objective of this study was to use these bench-top assays to examine S. galacifolia for antitumor, estrogen-receptor modulating and antimutagenic characteristics.

Plant materials
Shortia specimens (leaves and rhizome) were collected with permission from native populations in Oconee County (

Extraction
Shortia samples were washed under running water for 5 min and allowed to airdry at room temperature overnight. Plants were divided into leaf material, new growth rhizome and mature rhizome and dried in an oven at 40 o C for 48 hr. Samples (5 g) were ground, extracted with 80% methanol, placed on a lateral shaker and mixed for 8 hr at room temperature (further details are provided in the Supplementary material). Samples were centrifuged and the pellet was re-extracted, evaporated to dryness, reconstituted with 100% ethanol or DMSO (1g/mL), filtered and stored in the dark at 25 o C.
Dried RRL extracts, 2 g equivalent, were suspended overnight in 3 mL hexane. Samples were then extracted with 3 mL of 80% methanol, and centrifuged at 1800 g for 15 min. The hexane supernatant was removed and extracted again with 80% methanol.
Methanolic fractions were combined, filtered with 0.45 μm Acrodisc® and evaporated to dryness on a heated DriBath (45 o C) under a gentle stream of filtered air. Extracts were reconstituted to a concentration of 1g original RRL /ml 100% ethanol.

Antitumor assay
Antitumor activity of Shortia extracts was examined by using the TiA method detailed by McLaughlin's laboratory (McLaughlin et al., 1998). Preliminary experiments on hexane and methanol solvent suitability with Agrobacterium tumor induction experiment, were conducted (data not shown). Samples were reconstituted and diluted in DMSO for assay testing. Further details on the methodology are presented in the Supplementary material.
Shortia extracts (1 g/ml) were diluted in 50% ethanol to obtain four concentrations: 10 mg/ml (1:100), 1 mg/ml (1:1000), 0.1 mg/ml (1:10,000), and 0.01 mg/ml (1:100,000). Each tissue and dilution was tested by use of two separate culture dishes with twelve potato discs each for a total of 24 replicates per treatment. Three controls were used: (1) positive A. tumefaciens control in which no test sample was added, (2) a positive inhibitory control in which camptothecin was added and (3) a solvent control which contained 50% ethanol but no A tumefaciens. Discs contaminated with observable bacterial or fungal growth were not counted. Experiments were repeated three times and data were analyzed for statistical differences by using analysis of variance (ANOVA, SAS Institute; Cary, NC).

Antimutagenic assay
Antimutagenic characteristics of leaf, new growth rhizome and mature rhizome extracts were determined by using the Ames Salmonella/microsome mutagenic test with modifications (Mortelmans and Zeiger, 2000). Extracted plant samples were diluted 1:1000 in DMSO.
Cells were diluted 1:10 immediately before using in the tests. The assay consisted of combining controls or test compounds, the Salmonella tester strain, the mutagen 2-aminoanthracene (2-AA) and S9 liver microsomes in a soft top agar which was poured onto a minimal agar plate lacking histidine (see Supplementary material for further details). The S9 portions had one of the following added: 10.4 μl DMSO, 10.4 μl leaf extract, 10.4 μl new growth rhizome extract or 10.4 μl mature rhizome extract. One aliquot of the duplicate was treated with 6 μl (1 mg/ml) of the mutagen 2-aminoanthracene (2-AA) and the second had 6 μl DMSO. The DMSO-treated aliquot served as a control for each treatment. All aliquots were incubated for 30 min at 37 o C. After 48 hr, revertant colonies on control and test plates were counted (Figure 2). Triplicate plates were made for each treatment in three separate experiments for a total of 72 plates. Data were analyzed by ANOVA (SAS Institute, Cary, NC).
For study of the mutagenic/antimutagenic activity of Shortia crude extracts, UV-C (254 nm) radiation exposure as the mutagen or positive control for bacterial mutation was used (see Supplementary material for further details).

Statistical analysis
Analysis was conducted using ANOVA on SAS for the antitumor and antimutagenic assays and StatLIA for the estrogen receptor binding assay, respectively.

Antitumor assay
The mean tumors/disc for the control was 7.23±3.48. The positive inhibitory treatment with camptothecin treatment resulted in 98.2% inhibition of tumors, and solvent treatment alone resulted in 0.19±1.2 tumors/disc. All concentrations of extracts significantly (p=0.05) inhibited A. tumefaciens-induced tumor formation on potato discs when compared to controls with no extract (Table 2). For the leaf extract, 1:10,000 dilution significantly inhibited tumor initiation when compared to 1:100,000 dilution. For all extract types, 1:100 dilutions inhibited tumor formation better than 1:100,000 dilutions. Overall, the mature rhizome extract inhibited tumor initiation better than leaf or new growth rhizome extracts (Table 3). Table 1. Chemosystematic analysis of chemicals in members of Diapensiaceae.

Quercetin
Galax urceolata (Soltis et al., 1983) Spinasterol Berneuxia thibetica (Wang et al., 1998)  The preliminary assays, using either hexane or 80% methanol rhizome extracts of Shortia in the tumor induction assay, revealed that the percentage of tumor inhibition using a hexane extraction of rhizome was similar to that of methanol (data not shown).

Antimutagenic assay
Antimutagenic activity was evident in the Ames Salmonella/microsome assay for the crude extracts of Shortia leaf, new rhizome and mature growth rhizome (Table  4). All three plant extracts significantly inhibited the formation of histidineindependent revertant colonies after exposure to the mutagen 2-AA. In terms of inhibiting revertant colony formation, the new growth rhizome extract was slightly more active (p=0.03) than leaf extract, but not different from mature rhizome extract. There were no differences in the numbers of colonies formed in the DMSO control cells with no 2-AA added and the extracttreated cells with no mutagen added, indicating that the extracts expressed no mutagenic activity in the Ames Salmonella/microsome assay.
Neither mature rhizome extract nor DMSO controltreated cells activated the 2-AA mutagenic changes in the bacterial cells without the S9 mix, suggesting that the buffers and extracts alone were incapable of activating the 2-AA or mutating the cells (Table 5). Plant extracts had no effects on UV-Ctreated S. typhimurium cells (Table 6). There were no significant differences in revertant bacterial CFUs formed from UV-C treated cells with or without plant extracts. Cells treated with plant extracts but not UV-C, produced the same numbers of revertant colonies as controls with no extract.

Estrogen Receptor Binding Assay
Dilutions of the sample extracts were used to obtain the concentration or estrogen binding equivalents that displaced approximately 50% of 3 H-E2 from receptors.
The value for EBE was determined from an E2 standard curve and adjusted for concentration/g of original plant material. In the assay for ERα, a greater affinity was shown for RRL than for Shortia which was non-detectable (see Table 7). However, ERβ displayed greater affinity for mature Shortia rhizome>Shortia leaf>Shortia new growth rhizome>>RRL. (+) signifies % increase above the control **% Inhibition=1-(A-B) x 100/ (A-C) A=# of histidine revertants formed with UV-C in control B=# of histidine revertants formed with UV-C in the presence of extract C=# of revertants formed without UV-C or extract in the control ***Not applicable

Discussion
In modern medicine, plants are used as direct therapeutic agents, as raw materials for development of new synthetic products, and as markers for new compounds (Badal et al., 2017). Some useful plant-based pharmaceuticals have been derived from random, serendipitous screening of plants, such as the anticancer drugs Taxol from the Pacific yew tree, Taxus brevifolia, and camptothecin from the Chinese ornamental tree, Camptotheca acuminata (Badal et al., 2017;Shields, 2017). A chemosystematic approach can be used to supplement ethnobotanical knowledge and perhaps facilitate the selection of plants for screening (Badal et al., 2017;Larsson, 2007).
Rapid bench-top assays can be useful as initial, rapid methods to screen plant extracts for bioactivity. The TiA is one such assay developed based on the inhibition of Agrobacterium tumefaciens tumor formation on potato discs. Although Shortia has limited ethnobotanical history, it shares bioactive chemicals with sister genera in both the Eastern and Western hemispheres ( Table 1) that do have ethnomedicinal lineage (Hamel and Chiltoskey, 1975;Wang et al., 1998). Methanol extracts of Shortia leaf and rhizome showed inhibition of Agrobacterium tumor induction at several dilutions. A concentration of 100 μg/ml was chosen for initial screening of plant compounds for bioactivity (Boyd, 1997). At this dilution, Shortia leaf, new growth rhizome and mature rhizome extracts showed 74.5, 66.7 and 78.8% inhibition, respectively. The significant antitumor activity detected suggests that further investigations into the antitumor properties of Shortia are needed.
The Ames Salmonella/microsome mutagenic assay revealed that extracts of Shortia (100 μg/ml) have no mutagenicity to the TA100 strain of S. typhimurium. Chemicals such as the aromatic amines and hydrocarbons are biologically inactive until metabolized via the cytochrome-based P450 systems (Friedman and Smith, 1984). Shortia extracts, however, showed no increase in the revertant colonies after treatment with human liver homogenate (S9 mix), indicating a lack of substrates that could be oxidized by the metabolic activation system to form mutagenic metabolites.
In contrast, the extracts significantly inhibited S9-activated 2-AA bacterial mutagenesis, indicating that extracts were antimutagenic in the Ames test. Further analysis on the composition of Shortia may reveal which chemical(s) contributes to the antimutagenicity as the activity is different from that of kaempferol and quercetin, as these chemicals can be mutagenic for TA100, whereas ellagic acid generally remains antimutagenic (Friedman and Smith, 1984;Resende et al., 2012;Silva et al., 1997).
A benefit of using the recombinant ER binding assays as opposed to cell proliferation assays and estrogen response element (ERE) regulated reporter gene assays, is that it abrogates the need for timeconsuming maintenance of live animals or cell lines (Lackey et al., 2001). Additionally, these cellular methods may not specify which receptor isoform is expressed in the system nor take into account non-genomic or crosstalk effects of phytoestrogens in a cellular system (Lackey et al., 2001). The use of recombinant ERα and ERβ in receptor binding assays offers an inexpensive, rapid technique for screening compounds for potential estrogen receptor modulatory activity. Leaf and rhizome extracts from S. galacifolia displayed greater binding to ERβ than for ERα, and this behavior appears to be different than that of ellagic acid, which is present in RRL and chemotaxonomic analysis (Table 1) revealed was in Diapensiaceae (Harborne and Williams, 1973). The behavior of kaempferol, also present in Diapensiaceae, appears similar to the ER binding results exhibited by Shortia, and was implicated as contributing to ERβ binding activities found in red wine (Zoechling et al., 2009). However, the results from the antimutagenic assays indicate that kaempferol may not be acting exclusively, as the S9 microsomes increase the biotransformation to quercetin (Silva et al., 1997). The ER modulatory activity of Shortia also appears different than that of spinasterol which is present in Berneuxia (Jeon et al., 2005;Wang et al., 1998).
In summary, based on ethnobotanical history and chemosystematic analysis, the antitumor, ER binding and antimutagenic activities of extracts of the rare plant, S. galacifolia, were examined. These results warrant further investigation into the nature of the potential anticancer activity of the plant and indicate that a combination of ethnopharmacological and chemosystematic approaches may aid in screening plant materials in the search and development of new medicines or treatments.